The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2
The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2
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DOI：
10.1074/jbc.273.49.32554
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发表时间：
1998-12-04
影响因子：
4.8
通讯作者：
Ramponi, G
中科院分区：
文献类型：
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作者：
Caselli, A;Marzocchini, R;Ramponi, G
低分子量磷酸酪氨酸蛋白磷酸酶（LMW-PTP）与其它PTP没有一般的序列同源性，但其活性位点序列基序CXXXXXR和反应机制与所有PTP相同。LMW-PTP的主要功能是下调血小板衍生生长因子和胰岛素受体。竞争性抑制剂P-i保护酶不失活，表明H_2O_2反应是针对活性位点的。对失活酶进行的游离巯基分析表明，八个LMW-PTP半胱氨酸中只有两个被修饰。时程高效液相色谱-电喷雾质谱法，连同特定的放射性标记和胰蛋白酶指纹分析，使我们能够证明，过氧化氢导致Cys-12和Cys-17的氧化，形成二硫键。由于这两个残基都位于活性位点区域，这种修饰使酶失活，荧光光谱实验表明，在H2 O2氧化过程中，酶的折叠被修饰。由于生理浓度的H2 O2产生酶失活，并考虑到通过用低分子量硫醇还原来恢复活性，我们建议氧化应激条件和产生过氧化氢的其他过程调节细胞中的LMW-PTP。
Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs, The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors, Both human LMW-PTP isoenzymes are inactivated by H2O2. The enzymes are protected from inactivation by P-i, a competitive inhibitor, suggesting that the H2O2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H2O2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme, Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H2O2. Because a physiological concentration of H2O2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.